Arabidopsis thaliana H
MA2 is a Zn
2+ transporting P
1B-type ATPase required for
maintainingplant
metal ho
meostasis. HMA2 and all eukaryote Zn
2+-ATPases have unique conserved N- and C-ter
minalsequences that differentiate the
m fro
m other P
1B-type ATPases. Ho
mology
modeling and structuralco
mparison by circular dichrois
m indicate that the 75 a
mino acid long HMA2 N-ter
minus shares the
mages/gifchars/beta2.gif" BORDER=0 ALIGN="
middle">
mages/gifchars/alpha.gif" BORDER=0>
mages/gifchars/beta2.gif" BORDER=0 ALIGN="
middle">
mages/gifchars/beta2.gif" BORDER=0 ALIGN="
middle">
mages/gifchars/alpha.gif" BORDER=0> folding present in
most P
1B-type ATPase N-ter
minal
metal binding do
mains (N-MBDs). However,the characteristic
metal binding sequence CysXXCys is replaced by Cys17CysXXGlu21, a sequence presentin all plant Zn
2+-ATPases. The isolated HMA2 N-MBD frag
ment binds a single Zn
2+ (
Kd 0.18
mages/entities/
mgr.gif">M),Cd
2+ (
Kd 0.27
mages/entities/
mgr.gif">M), or, with less affinity, Cu
+ (
Kd 13
mages/entities/
mgr.gif">M). Mutagenesis studies indicate that Cys17,Cys18, and Glu21 participate in Zn
2+ and Cd
2+ coordination, while Cys17 and Glu21, but not Cys18, arerequired for Cu
+ binding. Interestingly, the Glu21Cys
mutation that generates a CysCysXXCys site isunable to bind Zn
2+ or Cd
2+ but it binds Cu
+ with affinity (
Kd 1
mages/entities/
mgr.gif">M) higher than wild type N-MBD.Truncated HMA2 lacking the N-MBD showed reduced ATPase activity without significant changes in
metal binding to trans
me
mbrane
metal binding sites. Likewise, ATPase activity of HMA2 carrying
mutations Cys17Ala, Cys18Ala, and Glu21Ala/Cys was also reduced but showed a
metal dependencesi
milar to the wild type enzy
me. These observations suggest that plant Zn
2+-ATPase N-MBDs have afolding and function si
milar to Cu
+-ATPase N-MBDs. However, the unique Zn
2+ coordination via twothiols and a carboxyl group provides selective binding of the activating
metals to these regulatory do
mains.Metal binding through these side chains, although found in different sequences, appears as a co
mmonfeature of both bacterial and eukaryotic Zn
2+-ATPase N-MBDs.